Selenoprotein p (Se-P) is a complex extracellular selenoprotein that is expressed by many tissues. Its cDNA implies that it contains 10 selenocysteine residues and that it is rich in cysteine and histidine. It is a glycoprotein. Se-P accounts for most of the selenium in rat plasma and its concentration fails in selenium deficiency. In the present grant period we have shown that isoforms of Se-P are present in plasma and have characterized 2 of them. Both appear to arise from the same mRNA. One is the full-length translation product of the mRNA and the other terminates at the second in-frame UGA, yielding a short isoform containing only 1 selenocysteine. Using immunohistochemistry, we have shown that Se-P binds to endothelial cells in liver, kidney, and brain. Diquat in low doses causes lipid peroxidation and liver necrosis in selenium-deficient rats. Injection of selenium protects against this injury at a time when Se-P has appeared but glutathione peroxidase has not. A time- course study of the model using electron microscopy demonstrates that the first cells injured are endothelial cells in the centrilobular regions. Thus, our collective evidence suggests that Se-P protects endothelial cells against oxidative injury. We propose to continue studying Se-P structure and activities with the aim of determining its biological functions. We will use chemical modification and mass spectrometry to determine the number of redox forms and true isoforms of Se-P, to determine the heparin-binding site(s) and the effect of heparin binding on configuration of Se-P, and to characterize the cysteines/selenols in Se-P. Knockout mice will be produced in which all forms of Se-P are deleted or the long isoform is deleted but the short isoform continues to be expressed. The mice will be studied to determine whether their phenotype involves endothelial cell damage. Cultured endothelial cells will be studied to assess binding of Se-P to them and protection of the cells by Se-P from oxidant substances. These and other studies are proposed to evaluate the role of Se-P in protecting endothelial cells (and other cells) against oxidant substances in the extracellular space.